Applications Link; This link will go to a common page for all
groups describing why we did these projects. (Do not work on this for
each group, we'll take care of it.
The above scheme uses bacterial cell-cell signalling
to synchronise repressilators among neighbouring bacteria (E.coli).
The repressilator is based on the Elowitz repressilator but is not identcal
in terms of the order of the repressors. In other words, if the Elowitz
oscillator is counter-clockwise, our design is clockwise. The Vibrio
fischeri LuxI-LuxR quorum sensing circuit components were particularly
suited because of their simplicity and also as they have been shown
to work in E.coli at the temperature range the repressilator functions
in. The magenta squiggles represent the possible use of one of two alternative
promoters for the tetR and luxI genes. This leads to four alternative
designs each with its own pros and cons. Simulations carried out by
our group did not help us in choosing any one particular design over
another. So we have proposed a strategy to synthesise the four combinations.
Even before assembly: Do all the parts work?
It may be worthwhile to test individual RBS, promoters, repressors
and reportes before assembling the parts as most of them have been
modified to meet the biobricks format and also because it is possible
that we do not know enough about their biology. A standard reporter
can be chosen to measure the function of all the parts so their their
relative strengths can be compared.
Testing the repressilator independent of the synchronisation
The Elowitz repressilator design has not been tested for different
configurations of repressors. We suggest that our design of the synchrollator
be tested independently of the quorum sensing synchronising circuit
for its repressilator-like behaviour(the design is modular to facilitate
this). At the next stage, various synchronising strategies could be
tried out. Tuning the RBS parts are a good way to "tune"
the repressilator till it works in the current configuration.
HSL (the autoinducer) is commercially available in pure form and
will prove useful in debugging the synchronisation module.
Testing Intercellular Communication
In order to test whether the homoserine-lactone (HSL) autoinducer
(AI) diffuses between the cells, one can make a HSL-dependent reporter
cell. In the scheme below, HSL is produced in the synchronized repressilator.
The reporter cell contains all the lux genes except luxI, which is
responsible for HSL production. In the lux system, bioluminescence
is promoted by the activated luxR-AI complex. However, since the reporter
cell lacks the ability to produce the HSL autoinducer, bioluminescence
by the reporter cell is evidence that the HSL is indeed diffusing
In order that the coculture of the two cell strains (reporter strain
and the repressilator strain) be sustained, we could engineer them
each degrades one of two antibiotics and culture them in presence
of both antibiotics. Both strains would be needed to deplete the antibiotics
from the medium. Other strategies such as each strain makes one of
two essential amino acids that the other cannot make could also be
Degradation of acyl-homoserine lactone
The half life of HSL is 24 hrs at pH of 7.5 and it is important that
a degradation mechanism that is faster than the desired period of
oscillations be introduced into the system. Cyclical degration of
HSL would generate better synchronisation signals than a constant
degradation mechanism. Possible degradation mechanisms that were considered
1. Flow of medium through the filter that washes away the HSL secreted
into the medium at a constant rate.
2. aiiA is an enzyme that inactivates acyl-HSL,
presumably by cleaving the acyl side-chain. aiiA
could be placed under the control of a tetR repressible promoter or
a lacI repressible promoter.
Placing aiiA under the control of lacI repressible
promoter seemed to be the best design as per our simulations. However,
it may be worthwhile to try other possibilities.
It may be necessary to test if the LVA-tagged aiiA enzyme is functional.
This can be done by introducing both the LuxR-LuxICDABE bioluminescence
cassette and the aiiA gene in E. coli and measuring the density at
which bioluminescence is observed.
List of known inhibitors of the system components
The following compounds can be used to debug the system by perturbing
one component of the system at a time.
anhydrotetracycline - inhibits tetR repressor
IPTG - inhibits lacI repressor
furanones - degrade LuxR activator. These compounds are synthesised
by a marine alga Delisea pulchra and the genes that synthesise
them are not known yet. They can be isolated from Delisea pulchra.
They bind LuxR and degrade it in a protease independent manner.